A tumor-suppressive Escherichia coli 18 and its uses

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a tumor-suppressive *Escherichia coli* 18 and its uses, and more specifically to *Escherichia coli* HZ_Ec_18 and its applications. This invention obtains multiple strains with potential to influence the tumor microenvironment through in vitro culture and purification of bacteria from cervical tissue and vaginal swabs of cervical cancer patients. Among them, *Escherichia coli* HZ_Ec_18 exhibits stronger tumor cell inhibitory activity and interaction with host cells, and can be used for the development of anti-tumor drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a tumor-suppressive Escherichia coli 18 and its uses, and more specifically to an Escherichia coli HZ_Ec_18 and its uses. Background Technology

[0002] Cervical cancer is a malignant tumor that occurs in the cervix and is the fourth most common malignant tumor among women worldwide. The main clinical manifestations of cervical cancer patients are heavy or abnormal vaginal bleeding, especially after sexual intercourse. Some patients may experience watery, mucous, or foul-smelling vaginal discharge. Advanced patients may experience lower limb edema, flank pain, and pain in the pelvis or lower back.

[0003] Cervical cancer is mainly classified into squamous cell carcinoma, adenocarcinoma, and adenosquamous carcinoma. Squamous cell carcinoma is a cancer caused by the malignant transformation of squamous epithelial cells; adenocarcinoma develops from subcolumnar reserve cells of the endometrium, simultaneously differentiating into glandular and squamous cells. Adenocarcinoma ranks second in incidence among cervical cancers globally, and its incidence is increasing year by year, with a trend towards affecting younger populations. Compared to squamous cell carcinoma, adenocarcinoma exhibits multiple pathological types, different cell morphologies, lower sensitivity to radiotherapy and chemotherapy, a higher recurrence rate, a greater tendency for distant metastasis, and significant adverse prognostic effects. However, pathological classification alone cannot adequately guide the treatment of adenocarcinoma patients. Currently, treatment plans for cervical adenocarcinoma still refer to those for squamous cell carcinoma, leading to overtreatment in some adenocarcinoma patients and causing various negative effects, complications, and functional impairments.

[0004] Currently, treatments for many cancers, including cervical cancer, primarily include traditional therapies (such as surgery, chemotherapy, and radiotherapy), targeted therapy, immunotherapy (such as immune checkpoint inhibitors and CAR-T cell therapy), gene therapy, and combination therapy strategies. However, these treatment options still face numerous challenges. For example, CAR-T therapy has limitations: in solid tumors, CAR-T cells struggle to effectively infiltrate the tumor microenvironment and are susceptible to immunosuppression; furthermore, traditional CAR-T preparation is costly and may cause side effects such as cytokine release syndrome (CRS). Another example is immunotherapy resistance: some patients do not respond to immune checkpoint inhibitors (such as PD-1 / PD-L1 inhibitors), possibly related to CD8-positive T cell depletion or metabolic disorders (such as glucose deprivation) in the tumor microenvironment. Furthermore, the applicability of targeted therapy is limited: for instance, KRAS mutations have long been considered "undruggable" targets, and despite advancements in next-generation inhibitors (such as D3S-001), some patients may still develop resistance. Furthermore, there is a need to optimize combination therapies: how to balance efficacy and toxicity (such as the potential side effects of combining ferroptosis inducers with CAR-T), and how to accurately screen biomarkers (such as PD-L1 and TMB) to predict treatment response remain unsolved problems.

[0005] Cervical cancer is closely related to persistent infection with high-risk human papillomavirus (HPV), especially HPV types 16 and 18. HPV oncogenic proteins E6 and E7 target p53 and Rb proteins, leading to uncontrolled cell proliferation and genomic instability, and inducing abnormal host DNA methylation or histone modifications. Squamous cell carcinoma is generally considered highly associated with HPV 16, while adenocarcinoma is more closely related to HPV 18. However, HPV infection alone is insufficient to cause cancer; other factors such as immunosuppression (e.g., HIV infection), long-term use of oral contraceptives, multiple pregnancies, and early sexual activity can also promote cervical cancer. Therefore, the main clinical treatment options for early-stage cervical cancer include hysterectomy, lymph node dissection, radiotherapy, and chemotherapy. Advanced-stage patients receive comprehensive treatment primarily based on radiotherapy, supplemented with chemotherapy drugs such as cisplatin and etoposide.

[0006] Recent research indicates that alterations to the tumor microenvironment are also a key factor influencing tumor development. Endogenous microbes, acting as a double-edged sword, truly participate in the tumorigenesis and progression process. During their growth, the microbial community secretes various metabolites such as lactic acid, short-chain fatty acids (butyric acid), secondary bile acids, and even bacterial toxins, all of which can alter the tumor microenvironment and exert diverse effects on tumor cells. These effects include promoting cancer cell proliferation and metastasis, suppressing the host immune response, and causing tumor cell immune escape. While the cervix was traditionally considered a sterile environment, recent research using high-throughput sequencing technology has confirmed the existence of a unique microbial community within cervical cancer tissue, including bacteria, fungi, and viruses. It has been reported that the development or metastasis of more than 16% of cancers worldwide (such as lung cancer, cervical cancer, breast cancer, and pancreatic cancer) is related to microorganisms. Researchers can identify a variety of microorganisms in different types of tumors, and the composition of these microorganisms is also affected by a variety of factors, resulting in significant differences between individuals, such as gender, age, physical condition, and daily habits (smoking and drinking). With the development of microbiome and culture omics, researchers can conduct in vitro culture and research on endogenous bacteria or fungi in tumor tissues based on multi-omics, making it possible to explore new targets for cancer treatment and tumor treatment intervention strategies.

[0007] Existing research also shows that by modifying viral vectors, bacterial delivery systems or natural strains to activate immune responses or direct killing mechanisms, most studies have shown that microorganisms are used as carriers to inhibit tumor cell activity. Related microbial therapies have also been studied in animal and clinical models, and have also shown significant effects in inhibiting tumor growth. This suggests that microbial intervention (such as antibiotic regulation or probiotic combination therapy) may become an important strategy and intervention method for exploring new cancer therapies.

[0008] Liu et al. published a study in Cancer Cell on Aspergillus polypolysaccharides derived from lung cancer tissue.Aspergillus sydowii It can induce MDSC infiltration and suppress the host immune response, thereby promoting cancer development. Cai et al. published a study in Cell revealing that intratumoral flora can alter the cancer cell skeleton, enhancing its resistance to mechanical shear forces in the blood, further promoting cancer cell metastasis to the lungs. Lauren et al. also reported in Cancer Cell that inert L-lactate-producing lactobacilli were found in cervical cancer patient tissues. Lactobacillus iners This bacterium is significantly associated with decreased patient survival rates; it can induce metabolic rearrangement in cervical cancer cells, enhancing their resistance to radiotherapy and chemotherapy. *E. coli* carrying the PKS gene (encoding a DNA damage toxin) E. coli After adhering to host cells, it secretes toxic proteins that cause microsatellite instability and chromosomal damage in host immune cells, driving the development of colorectal cancer. Klebsiella pneumoniae Klebsiella pneumoniae It can activate TLR4 by expressing the penicillin-binding protein PBP1B, promote the proliferation of liver cancer cells, and activate pro-cancer signaling pathways, ultimately leading to liver cancer progression. Barath et al. discovered *Fusobacterium nucleatum*... Fusobacterium nucleatum It can adhere to pancreatic epithelial cells and stimulate tumor and normal cells to secrete granulocyte-macrophage colony-stimulating factors or chemokines, thereby enhancing the proliferation and invasion of cancer cells and further promoting the development of pancreatic cancer. It is evident that the human microbiome plays a crucial role in tumorigenesis and cancer treatment. Although the gut microbiome has always been a major focus of research in these fields, the understanding of the existence and pathological relevance of the tumor microbiome is beginning to emerge. Revealing the important mechanisms by which gut microbiota or endogenous tumor microorganisms promote cancer development or metastasis, and whether the origin of intratumoral microbiota is related to the gut microbiome, are important scientific questions that urgently need to be addressed in cancer treatment. Summary of the Invention

[0009] The purpose of this invention is to screen for a potential functional strain of bacteria that can be used as a drug or nanobody delivery system and has anti-tumor activity, and can be used for the treatment of cervical cancer, liver cancer or ovarian cancer.

[0010] A second objective of this invention is to provide applications of the aforementioned functional strains.

[0011] To address the aforementioned problems, this invention provides a tumor-suppressive Escherichia coli 18 strain, designated HZ_Ec_18, and classified as follows: Escherichia coli HZ_Ec_18 has been deposited at the China Center for Type Culture Collection, accession number: CCTCC No: M20251870.

[0012] The present invention also provides the use of the tumor-suppressive Escherichia coli 18 in the preparation of a drug having at least one of the following (1)-(3):

[0013] (1) Drugs for the prevention and / or treatment of tumors;

[0014] (2) Drugs with anti-cancer activity;

[0015] (3) Drugs with anti-pan-cancer activity.

[0016] Specifically, the tumors include cervical cancer, liver cancer, or ovarian cancer.

[0017] Specifically, the cervical cancer includes at least one of cervical adenocarcinoma, cervical squamous cell carcinoma, and cervical adenosquamous carcinoma.

[0018] Specifically, the cancer cells include cervical cancer cells HeLa, cervical cancer cells SiHa, human liver cancer cells HepG2, or human ovarian cancer cells A2780.

[0019] Specifically, the tumor suppressor Escherichia coli 18, namely Escherichia coli HZ_Ec_18, exists in the form of living cells.

[0020] Specifically, the number of live bacteria of the tumor-suppressive Escherichia coli 18, namely Escherichia coli HZ_Ec_18, is MOI=10-60. The number of live bacteria varies depending on the tumor cells. For example, MOI can be adjusted to 10, 20, 40 or 60.

[0021] Specifically, the tumor-suppressive Escherichia coli 18, namely Escherichia coli HZ_Ec_18, is the sole active ingredient.

[0022] The present invention also provides a pharmaceutical formulation comprising a therapeutically effective amount of the tumor-suppressive Escherichia coli 18, namely Escherichia coli HZ_Ec_18.

[0023] Specifically, the pharmaceutical formulation also includes other pharmaceutically acceptable carriers and / or excipients compatible with the Escherichia coli HZ_Ec_18.

[0024] Specifically, the pharmaceutical formulation is a pharmaceutically acceptable non-gastrointestinal and / or gastrointestinal dosage form; wherein,

[0025] The non-gastrointestinal drug delivery dosage forms include at least one of injection dosage forms, cavity dosage forms, mucosal dosage forms, and skin dosage forms;

[0026] The gastrointestinal dosage forms include at least one of tablets, granules, capsules, solutions, powders, sustained-release preparations, emulsions, suspensions, syrups, and drops.

[0027] In embodiments of the present invention, the pharmaceutical preparation may further include at least one of chemotherapy drugs, immune checkpoint inhibitors, immune cell therapy drugs, ferroptosis inducers, and KRAS inhibitors.

[0028] In a specific embodiment of the present invention, the chemotherapy drugs include at least one of cisplatin, etoposide, paclitaxel, camptothecin, 5-fluorouracil, doxorubicin, mitomycin, and epirubicin.

[0029] In a specific embodiment of the present invention, immune checkpoint inhibitors include inhibitors targeting at least one of PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG-3, TIGIT, VISTA, BTLA, CD27, CD28, CD70, CD80, CD86, CD137, CD276, KIRs, TNFRSF4, GITR, GITRL, 4-1BBL, A2aR, VTCN1, IDO, and KLRA.

[0030] In a specific embodiment of the present invention, the immune cell therapy drug includes at least one of T cell therapy drugs, tumor-infiltrating lymphocyte therapy drugs, and NK cell therapy drugs.

[0031] This invention obtained multiple bacterial strains with potential to influence the tumor microenvironment through in vitro culture and purification of bacteria from cervical tissue and vaginal swabs of cervical cancer patients. Among them, *Escherichia coli* HZ_Ec_18 exhibited stronger tumor cell inhibitory activity and host cell interaction compared to other *E. coli* strains, suggesting that its genes may have undergone large-scale recombination or mutations at key gene sites, leading to significant differences in activity compared to other strains. Furthermore, this invention used transmission electron microscopy to observe morphological changes in *E. coli* HZ_Ec_18 after co-incubation with cervical cancer tumor cells, revealing mitochondrial swelling, lysosomal disruption, intracellular degradation, and cell membrane damage.

[0032] This invention provides a novel "microbe-to-host" perspective on tumor treatment through the interaction between intratumoral microbiota and tumors. The complex interaction network between microbiota and tumors is driving the development of personalized and combination therapies, revealing the crucial role of microbiota in tumor development, metastasis, and treatment. Therefore, the *Escherichia coli* HZ_Ec_18 with antitumor activity isolated from clinical tumor samples in this invention provides important theoretical and material support for subsequent bacterial-based intervention therapies.

[0033] This invention involves the direct isolation and culture of bacterial strains in cervical cancer patient tissues, which can serve as potential functional strains for drug or nanobody delivery systems and antitumor activity.

[0034] Biological Preservation Instructions

[0035] Escherichia coli HZ_Ec_18, deposited on August 21, 2025, with accession number CCTCC NO: M20251870, and classified as Escherichia coli HZ_Ec_18. Escherichia coli HZ_Ec_18, the depository is China Center for Type Culture Collection, located at Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of this disclosure, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this disclosure. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0037] Figure 1 The results show the vaginal flora composition analysis of cervical cancer patients and healthy women; where A represents the results of LefSeq analysis and genus-level intratumoral flora composition analysis (HC: healthy women cohort, HCRD: cervical cancer patients cohort); B represents the KEGG database flora function annotation results.

[0038] Figure 2 Annotation results of vaginal species in cervical cancer patients and healthy women;

[0039] Figure 3 The results of the correlation analysis between vaginal flora and CRP in cervical cancer patients are presented. Among them, A represents the comparison analysis of serum CRP concentration between healthy women and cervical cancer patients (Wilcoxon rank-sum test, ***P=0.0003); B and E represent the results of correlation analysis (Pearson correlation analysis) between CRP and Enterobacteriaceae (B), Enterobacteriaceae (C), Escherichia coli-Shigella (D), and Staphylococcus spp. (E), respectively.

[0040] Figure 4 The results of Escherichia coli isolation and identification are shown. Among them, A is the morphological results of Escherichia coli observed by SEM, showing bacilli with a length of about 2 µm and bacterial flagella (scale bar is 2 µm); B is a phylogenetic tree constructed based on 16S rRNA gene sequences, showing that different strains can be clustered into different gene clusters (nearest neighbor method, bootstrap value = 2000).

[0041] Figure 5 The results of the effects of different strains of Escherichia coli on the growth of cervical cancer cells;

[0042] Figure 6The effects of different Escherichia coli on tumor cell activity were investigated; A represents the effect of Escherichia coli 18 on HeLa cells; B represents the effect of Escherichia coli 8 on SiHa cells.

[0043] Figure 7 The results show the effects of inactivated Escherichia coli and the supernatant after bacterial culture on the growth of cervical cancer cells; where AB represents the effect of inactivated bacterial strains on the activity of cervical cancer cells; and CD represents the effect of the supernatant after bacterial culture on the activity of cervical cancer cells.

[0044] Figure 8 The results show the effects of Escherichia coli on the activity of different tumor cells; where A and B represent the effects of Escherichia coli 18 on hepatocellular carcinoma cells (HepG2) and ovarian cancer cells (A2780), respectively.

[0045] Figure 9 The results show the lactate concentration after co-incubation of Escherichia coli with tumor cells; where A and B are the L-lactic acid (A) and D-lactic acid (B) concentrations in the co-culture systems of Escherichia coli with HeLa and SiHa, respectively. Detailed Implementation

[0046] The above description is merely an embodiment of this disclosure and is not intended to limit this disclosure. Any modifications, equivalent substitutions, improvements, etc., made within the principles of this disclosure should be included within the protection scope of this disclosure.

[0047] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings and specific examples.

[0048] In the following embodiments of the present invention, unless otherwise defined, all technical terms used have the same meaning as commonly understood by those skilled in the art.

[0049] This invention discloses an Escherichia coli strain, a pharmaceutical composition, and their applications. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0050] In the description of this invention, the list of items connected by the term "at least one of" or other similar terms may mean any combination of the listed items. For example, if items A and B are listed, then the phrase "at least one of A and B" means only A; only B; or A and B. In another example, if items A, B, and C are listed, then the phrase "at least one of A, B, and C" means only A; or only B; only C; A and B (excluding C); A and C (excluding B); B and C (excluding A); or all of A, B, and C. Item A may contain a single element or multiple elements. Item B may contain a single element or multiple elements. Item C may contain a single element or multiple elements.

[0051] Unless otherwise specified, the terms "comprising" and "including" as used in this application can be open-ended or closed-ended. For example, "comprising" and "including" can mean that other components not listed may also be included, or that only the listed components may be included.

[0052] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges or individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0053] The term "strain" refers to a member of a bacterial species that possesses genetic characteristics that distinguish it from closely related members of the same bacterial species. These genetic characteristics may include the complete or partial absence of at least one gene, the complete or partial absence of at least one regulatory region (e.g., promoter, terminator, riboswitch, ribosome binding site), the presence of at least one recombinant gene, the presence of at least one mutant gene, the presence of at least one exogenous gene (a gene from another species), the presence of at least one non-natural plasmid, the presence of at least one antibiotic resistance cassette, or a combination thereof. Genetic characteristics between different strains can be identified by PCR amplification, optionally followed by DNA sequencing of the genomic region of interest or the entire genome. In cases where a strain (compared to another strain of the same species) acquires or loses antibiotic resistance or acquires or loses biosynthetic capacity (e.g., auxotrophic strains), the strain or nutrient / metabolite can be distinguished by selection or anti-selection using antibiotics. It is known in the art that bacterial species can be classified and identified using conventional taxonomic methods and molecular biological methods. Conventional taxonomic methods include, for example, cell morphology observation, Gram staining, flagellar staining, various metabolic experiments, etc. Molecular biology methods include ribosomal RNA sequencing and whole-genome sequencing-based methods.

[0054] The term "immune checkpoint inhibitor" is an antagonist that targets an immune checkpoint protein. This immune checkpoint inhibitor enhances proteins that stimulate the immune response or blocks proteins that inhibit the immune response, thereby exhibiting an anti-cancer effect through the immune response. In some embodiments, the immune checkpoint inhibitor can be a protein or peptide, such as a soluble fusion protein; it can be an antibody or its antigen-binding fragment that binds to the immune checkpoint protein to be inhibited; or it can be an inhibitory nucleic acid (e.g., siRNA molecules, shRNA molecules, antisense RNA) that specifically binds to mRNA encoding the immune checkpoint protein.

[0055] In the following specific embodiments, operations without specified conditions are performed under standard conditions or conditions recommended by the manufacturer. Raw materials without specified manufacturers and specifications are all commercially available products.

[0056] In the following embodiments of the present invention, 16S rRNA sequencing was performed on bacteria from vaginal secretions of 30 healthy women and 63 cervical cancer patients to identify high-abundance bacterial strains in cervical cancer. Furthermore, fresh cervical tissue and vaginal secretions from 32 cervical cancer patients were collected and subjected to in vitro bacterial isolation and culture under anaerobic and aerobic conditions, respectively. By analyzing biochemical indicators of healthy and patient women, and based on the significantly elevated C-reactive protein (CRP, an immune-related indicator) in cancer patients, combined with 16S rRNA sequencing data, correlation analysis (Pearson analysis) was performed between the relative abundance of bacterial strains and CRP to screen for functional strains in cervical cancer patients that have the potential to regulate host immune signaling pathways.

[0057] In the following embodiments of the present invention, after sample collection, all sample processing, bacterial culture and purification were completed under sterile laboratory conditions. 16S rRNA sequencing and evolutionary analysis were used, and bacterial morphology was observed under a scanning electron microscope to complete bacterial identification. The effect of highly abundant culturable strains on tumor cell activity was detected using the CCK-8 kit method. Finally, the concentration of D- / L- lactate in the supernatant after co-culturing the strains and tumor cells was quantified using a colorimetric method, i.e., the effect of the strains on the tumor microenvironment.

[0058] After screening, the following embodiment of the present invention yielded a strain of Escherichia coli. E. coli _18, its strain number is HZ_Ec_18, and its classification name is Escherichia coli HZ_Ec_18 has been deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Bayi Road, Wuchang District, Wuhan, Hubei Province, China. The deposit date is August 21, 2025, and the accession number is CCTCCNo: M20251870.

[0059] In embodiments of the present invention, the *E. coli* strain may also be a variant of *E. coli* strain with accession number CCTCC No: M20251870. Any variant, mutant, recombinant, or derived strain obtained from HZ_Ec_18 as starting material through natural mutation, mutagenesis, screening, genetic engineering, gene editing, recombination technology, or other conventional molecular biology methods in the art, as long as it substantially retains or can achieve the same or similar tumor suppressive activity and usable uses as HZ_Ec_18, is considered to fall within the scope of protection claimed by the present invention. To eliminate ambiguity, the protection of the present invention can be determined based on either structural features or functional equivalence (i.e., maintaining tumor suppressive activity).

[0060] The Escherichia coli HZ_Ec_18 has been verified to have anti-cancer activity. For example, it has shown a certain inhibitory effect on cervical cancer cells HeLa, cervical cancer cells SiHa, human liver cancer cells HepG2, or human ovarian cancer cells A2780, i.e., anti-tumor activity.

[0061] Similarly, the following embodiments of the present invention further verify that the *Escherichia coli* HZ_Ec_18 has anti-pan-cancer cell activity.

[0062] Based on this, the Escherichia coli HZ_Ec_18 described in this invention has an inhibitory effect on tumors and can be used to prepare drugs for the prevention and / or treatment of tumors, such as drugs for the prevention or treatment of cervical cancer, liver cancer or ovarian cancer.

[0063] Furthermore, when the *E. coli* HZ_Ec_18 is used to prepare drugs for the prevention and / or treatment of tumors, the *E. coli* HZ_Ec_18 exists in the form of living cells. It is understood that, as an active ingredient in antitumor drugs, *E. coli* HZ_Ec_18 can exert a better therapeutic effect when it exists in the form of living cells.

[0064] In some specific applications, when the Escherichia coli HZ_Ec_18 is used to prepare drugs for the prevention and / or treatment of tumors, the viable count of the Escherichia coli HZ_Ec_18 is MOI = 10-60. Different MOI values ​​are selected according to the differences in cancer cells used, such as MOI = 10, 20, 40 or 60, or any range between the above values.

[0065] In some specific applications, the *Escherichia coli* HZ_Ec_18 can be used as the sole active ingredient in an antitumor drug; alternatively, it can be combined with other ingredients possessing antitumor activity known in the art. For example, it can be combined with drugs known in the art for the prevention or treatment of cervical cancer, liver cancer, or ovarian cancer.

[0066] The present invention also provides a pharmaceutical preparation comprising a therapeutically effective amount of the *Escherichia coli* HZ_Ec_18. It should be noted that the term "therapeutically effective amount" refers to the dosage of the drug required to produce an effective effect. This "therapeutically effective amount" can be adjusted and varied according to actual circumstances and is ultimately determined by medical personnel, taking into account factors such as the route of administration and the nature of the preparation, the recipient's weight, age, and other general characteristics, as well as the nature and severity of the disease being treated.

[0067] In some specific applications, the pharmaceutical formulation provided by this invention further includes other pharmaceutically acceptable carriers and / or excipients compatible with the *Escherichia coli* HZ_Ec_18. It should be noted that "other pharmaceuticals" here refers to drugs compatible with the *Escherichia coli* HZ_Ec_18 that do not cause inactivation, reduced activity, or hydrolysis of the *Escherichia coli* HZ_Ec_18, and that, after synergistic action with the *Escherichia coli* HZ_Ec_18, can enhance the therapeutic effect of the *Escherichia coli* HZ_Ec_18. The aforementioned carriers can be commonly used drug carriers in the art, such as chitosan, liposomes, alginate, agar, fibrin, collagen, and synthetic polymer carriers. The excipients mentioned above are physiologically inactive and do not affect the efficacy, content determination, or stability of the active pharmaceutical ingredient. Their main purpose is to facilitate the preparation and clinical application of the formulation. For example, they can be commonly used materials in this field such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, light magnesium oxide, calcium carbonate, dry starch, sodium hydroxymethyl starch, low-substituted hydroxypropyl cellulose, effervescent disintegrants, and crospovidone.

[0068] In some specific applications, the pharmaceutical preparations provided by this invention are pharmaceutically acceptable oral dosage forms. For example, powders, granules, pills, capsules, tablets, ointments, liquid preparations, gels, etc. Accordingly, depending on the actual dosage form, the administration method can be selected from inhalation, inhalation, nasal administration, sublingual administration, parenteral administration, etc.

[0069] Example 1

[0070] This embodiment is based on the 16S rRNA sequencing of bacteria in vaginal secretions and the analysis of bacterial species diversity in 30 healthy women and 63 cervical cancer patients.

[0071] Bacterial genomic DNA was extracted from samples using a kit-based method, and purity and concentration were detected using Nanodrop 2000. DNA fragmentation was performed using Covaris M 220, and fragments of approximately 400 bp were screened and enriched. Subsequently, PE libraries were constructed using a kit-based method, followed by PCR amplification and 16S rRNA sequencing. The raw sequencing data were quality controlled using Fastp and BWA software and compared with human DNA sequences to remove highly similar contaminating reads. MEGAHIT was used to assemble and splice the filtered sequences, screening contigs longer than 300 bp for ORF prediction. The predicted gene sequences were then clustered using CD-HIT software. Finally, SOA Paligner software was used to enrich and align high-quality reads and non-redundant genes for each sample, and the abundance of each gene in the corresponding samples was calculated. The DIAMOND online analysis tool was used to compare non-redundant gene sequences with the NR database to obtain annotation information for species at different taxonomic levels.

[0072] This embodiment is based on the completed 16S rRNA sequencing and diversity analysis of bacteria in vaginal secretions from 30 healthy women and 63 cervical cancer patients. The results are attached. Figures 1-2 As shown in the figure, the results indicate that the vaginal flora composition is disordered in patients with cervical cancer.

[0073] Figure 1 The Latin translations are shown in the table below.

[0074]

[0075] Figure 2 The Chinese Latin or English definitions are shown in the table below.

[0076]

[0077] As attached Figure 1 The LefSeq analysis results shown in Figure A indicate that Firmicutes, Clostridium, and Bacteroidetes are highly abundant flora in the vagina of cancer patients. At the class level, Staphylococcus, anaerobic cocci, and Corynebacterium are significantly enriched. There are significant differences in bacterial composition between the two groups. The relative abundance of beneficial bacteria such as Lactobacillus and Bifidobacterium is reduced in the tumor group, while the relative abundance of some opportunistic pathogens such as Proteus, Staphylococcus, and Streptococcus is increased. This suggests that tumor lesions cause changes in the vaginal environment, which also indicates that the disordered composition of the intratumoral flora in patients leads to changes in dissolved oxygen and pH in the tumor microenvironment.

[0078] As attached Figure 1The KEGG annotation results shown in A indicate that the functions of intratumoral bacteria are mainly enriched in the biodegradation and metabolism of exogenous substances such as amino acids, carbohydrates, energy metabolism, and membrane transport functions, which are speculated to be related to the energy supply for the excessive proliferation, invasion, or migration of tumor cells.

[0079] like Figure 2 The results of species annotation in the vagina of cervical cancer patients and healthy women are shown. The 16S rRNA sequencing results can detect strains with different abundances, such as Shigella, Fusobacterium, and Corynebacterium.

[0080] Example 2

[0081] This embodiment analyzes the correlation between intratumoral flora and C-reactive protein (CRP) in cervical cancer. By analyzing changes in patients' biochemical factors, Pearson analysis was used to perform correlation analysis between CRP and the relative abundance of various bacterial communities.

[0082] In this embodiment, biochemical factors in the serum of 63 cervical cancer patients and 30 healthy women were analyzed, such as... Figure 3 The results shown in Figure A indicate that C-reactive protein levels are significantly elevated in cervical cancer patients.

[0083] In this embodiment, the Pearson analysis method was used to perform correlation analysis between the relatively abundant bacterial communities and CRP concentrations in the two cohorts. For example... Figure 3 Results shown in Figures B, C, D, and E indicate that high CRP concentrations were positively correlated with the relative abundance of Enterobacteriaceae in the vagina of cervical cancer patients (R=0.2, P<0.1) and negatively correlated with Staphylococcus spp. (R=-0.099). Due to the small sample size, a new correlation analysis will be conducted with a larger sample size in the future. It is speculated that Enterobacteriaceae (Escherichia coli-Shigella) may activate the host immune response or regulate the tumor microenvironment, affecting cell activity.

[0084] Example 3

[0085] This embodiment describes the isolation and identification of Escherichia coli based on the intratumoral flora of cervical cancer.

[0086] In this embodiment, cervical tissue and vaginal secretions from cervical cancer patients were collected and pretreated under sterile laboratory conditions. The tissues were washed five times with sterile saline, homogenized, and spread onto Brain Heart Infusion (BHI) agar. Vaginal swabs were directly spread onto the same medium and incubated at 37°C for 24 hours. Samples under anaerobic conditions underwent the same treatment. The bacterial strain was purified through 3-4 consecutive cultures. Single bacterial clones were picked and cultured in BHI liquid medium until the logarithmic growth phase for preservation, and then frozen at -80°C. After resuscitation, genomic DNA was extracted from the bacteria. The 16S rRNA gene was amplified using 27F and 1492R primers and sequenced. Phylogenetic analysis was performed using NCBI tools and MEGA software to preliminarily identify the strain.

[0087] In this embodiment, bacterial in vitro culture was performed on vaginal secretions and cervical tissues collected from 32 cervical cancer patients, and a total of 18 strains of Escherichia coli were isolated and cultured.

[0088] Escherichia coli strain 8 from 18 strains was selected and cultured in BHI liquid medium for 3-4 hours (37℃, 180 rpm) until the logarithmic growth phase (OD600 value of 0.5). The bacterial cells were then recovered (5000g, 3min). The supernatant medium was discarded, and the sample was washed three times with phosphate-buffered saline (PBS). The sample was then fixed overnight with 2.5% glutaraldehyde solution at 4℃. After treatment with alcohol gradient dehydration, critical point drying, and gold sputtering, the bacterial morphology was observed under a scanning electron microscope. All samples were imaged using a field emission scanning electron microscope (JOEL).

[0089] This embodiment uses SEM to observe the morphology of Escherichia coli No. 8. The bacteria have flagella and are 1-2 µm long (e.g., Figure 4 (A) Using 16S rRNA sequencing and evolutionary analysis, Escherichia coli was identified. The results showed that multiple copies of genes encoding 16S rRNA exist in the E. coli genome (e.g., Figure 4 (See Figure B). As can be seen, the 18 Escherichia coli strains screened in this embodiment mainly clustered into 4 groups, and their genes were highly consistent with the Escherichia coli reference strains in the NCBI database. Based on the above methods, the isolation, purification and identification of Escherichia coli were completed.

[0090] Example 4

[0091] This embodiment further performs preliminary in vitro antitumor activity tests on 18 selected Escherichia coli strains (targeting two types of cervical cancer tumor cells).

[0092] Escherichia coli (18 strains and DH5α control strain) frozen at -80℃ were streaked onto BHI agar medium and incubated at 37℃ for 16-18 hours. Single colonies were picked and inoculated into BHI broth medium and cultured to the logarithmic growth phase (37℃, 180 rpm). The bacterial cells were then recovered by centrifugation (7000g, 5 min), washed three times with sterile PBS solution, and resuspended in sterile PBS solution for subsequent co-culture with cells.

[0093] One day before the experiment, administer 1×10 4HeLa and SiHa cells were seeded into 96-well plates at a cell / mL ratio, and the cell supernatant was discarded. 100 μL of DMEM medium was added to each well. The effect of the bacterial strain on tumor cell viability was initially assessed at MOI=50. The effect of bacterial infection dose on tumor cell viability (dose-dependent) was verified at MOIs of 10, 20, 40, and 60, with a mock control, PBS group, and bacterial control group (DH5α Escherichia coli) established. After 1 h in a cell culture incubator, the cells were washed three times with PBS buffer, and 100 μL of DMEM medium containing 2% FBS and 2% PS was added to each well. The cells were incubated for 24 h, then the medium was discarded, and 100 μL of fresh cell culture medium containing 10% CCK-8 was added. The cells were incubated at 37°C for 1-2 h, and the absorbance at 450 nm was measured to detect the effect of the bacteria on tumor cell viability. Cell viability was calculated and analyzed (CCK-8 method).

[0094] like Figure 5 The results show the effects of co-culturing 18 viable *E. coli* strains selected in this invention with SiHa. Specifically, the 18 viable *E. coli* strains were co-cultured with SiHa (MOI=50), and the cell viability after treatment was detected using the CCK-8 assay. The results indicate that *E. coli* strains 8, 11, 15, and 18 can inhibit the activity of SiHa. Strain 18 showed the strongest inhibition of tumor cell activity, with an inhibition rate of 90%-100%. Strains 11 and 15 inhibited SiHa survival by approximately 50%.

[0095] Further analysis of strain 18 was conducted to determine its antitumor activity and its effects on HeLa and SiHa cells. Different bacterial infection doses of 10 CFU, 20 CFU, 40 CFU, and 60 CFU were used. (See attached image) Figure 6 The results shown in Figures A and B indicate that the antitumor activity of Escherichia coli No. 18 was dose-dependent on the bacterial infection dose.

[0096] This embodiment further tested the effect of inactivated Escherichia coli and bacterial culture supernatant on the activity of cervical cancer tumor cells. The CCK8 assay was used to determine the activity of inactivated Escherichia coli (… E. coli _1、 E. coli _5、 E. coli _8、 E. coli _11、 E. coli The effects of _18) and the supernatant after bacterial culture on the growth of cervical cancer cells (HeLa and SiHa) were as follows: Figure 7 As shown. It can be seen that using inactivated strains (such as...) Figure 7 (A and B) and bacterial culture supernatant (e.g. Figure 7 C and D) do not affect tumor cell activity.

[0097] Example 5

[0098] This embodiment further tests the screened E. coli. E. coli Effects of _18 on the activity of different tumor cells. Following the cell co-culture method described in Example 4 above, Escherichia coli (18 strains and DH5α control strain) frozen at -80 ℃ were streaked onto BHI agar medium and cultured at 37 ℃ for 16-18 h. Single colonies were picked and inoculated into BHI broth medium and cultured to the logarithmic growth phase (37 ℃, 180 rpm). The cells were then centrifuged (7000 g, 5 min), washed three times with sterile PBS solution, and resuspended in sterile PBS solution for subsequent co-culture with cells.

[0099] One day before the experiment, administer 1×10 4 Human hepatocellular carcinoma HepG2 cells and human ovarian cancer A2780 cells were seeded into 96-well plates at a cell / mL ratio, respectively. The cell supernatant was discarded, and 100 μL of DMEM medium was added to each well. The effect of the bacterial strain on tumor cell viability was initially detected at an MOI of 50. The effect of the bacterial infection dose on tumor cell viability (dose-dependent) was verified at MOIs of 10, 20, 40, and 60, with a mock control, a PBS group, and a bacterial control group (DH5α Escherichia coli). After 1 h in a cell culture incubator, the cells were washed three times with PBS buffer, and 100 μL of DMEM medium containing 2% FBS and 2% PS was added to each well. The cells were incubated for 24 h, then the medium was discarded, and 100 μL of fresh cell culture medium containing 10% CCK-8 was added. The cells were incubated at 37°C for 1-2 h, and the absorbance at 450 nm was measured to detect the effect of the bacteria on tumor cell viability. Cell viability was calculated and analyzed using the CCK-8 method.

[0100] Determination of Escherichia coli using the CCK8 assay E. coli The effect of _18 on the growth of human hepatocellular carcinoma cells (HepG2) and human ovarian cancer cells (A2780) was investigated, and the results are as follows: Figure 8 As shown.

[0101] It can be seen that the Escherichia coli mentioned E. coli _18 inhibited the activity of HepG2 cells, with the inhibition rate reaching a maximum of 35% at an MOI of 60 (P = 0.0001). E. coli _18 also significantly inhibited the activity of A2780 cells in a dose-dependent manner, with the inhibition rate reaching its maximum at an MOI of 60, reaching 45% (P < 0.0001).

[0102] The above results demonstrate that the *Escherichia coli* strains screened, isolated, and purified by this invention are effective. E. coli _18 has potential anti-pan-cancer activity and has a wider range of applications.

[0103] The E. coli screened in this embodiment E. coli _18, its strain number is HZ_Ec_18, and its classification name is Escherichia coli HZ_Ec_18 has been deposited at the China Center for Type Culture Collection, located at Wuhan University, Bayi Road, Wuchang District, Wuhan, Hubei Province, on August 21, 2025, with accession number CCTCC No: M20251870.

[0104] Example 6

[0105] During the co-culture of *E. coli* and tumor cells, the supernatant of the cell culture medium turned a deep yellow, suggesting that the metabolic processes of the bacteria or cells changed and secreted acidic substances during co-culture. Literature review indicates that D-lactic acid and L-lactic acid play important roles in tumor development. This embodiment uses a screened *E. coli* and tumor cell co-culture system to detect lactic acid content.

[0106] Eighteen selected strains of *E. coli* and DH5α *E. coli* were used to infect HeLa and SiHa cells with an MOI of 50, respectively. A mock control and a PBS control group were also included. After 5 hPE cycles, the supernatant was collected, and the lactate content in the supernatant was measured using an L-lactate assay kit (AATBioquest) and a D-lactate assay kit (AAT Bioquest). Standard curves were prepared according to the reagent manufacturers' instructions, and the samples were processed and analyzed using a Varioskan LUX microplate reader (Thermo).

[0107] L-lactic acid and D-lactic acid in the supernatant of tumor cells co-cultured with 18 strains of Escherichia coli and DH5α were detected using a kit. The results are shown in the attached figure. Figure 9 As shown in A and B.

[0108] It is evident that the concentrations of D- and L-lactic acid in the HeLa and SiHa systems co-cultured with different E. coli isolates were significantly different from those in the control group. The trends in acidic substances were consistent in both systems. E. coli _8, HZ_Ec_18 (P<0.0001), E. coli _11 and E. coli The L-lactic acid content in the supernatant after co-incubation with HeLa cells was significantly higher than that in other samples (P < 0.01). E. coli _11 and E. coli The D-lactic acid content in the supernatant of _15 (P < 0.0001) after co-incubation with SiHa cells was significantly higher than that in other samples; E. coli _8、 E. coli _11、 E. coli The D-lactic acid content in the supernatant of HZ_Ec_15 and HZ_Ec_18 (P<0.0001) after co-incubation with HeLa cells was significantly higher than that in other samples; E. coli _11、 E. coli The D-lactic acid content in the supernatant of HZ_Ec_18 and HZ_Ec_18 (P<0.0001) after co-incubation with SiHa cells was significantly higher than that of other samples. The interaction between HeLa and Escherichia coli was the most significant, and the change in lactic acid concentration was the most obvious. This indicates that after the interaction between the intratumoral flora and tumor cells, both bacteria and tumor cells can affect the tumor microenvironment through metabolic reprogramming, and ultimately affect tumor development.

[0109] In summary, this invention, through 16S rRNA sequencing diversity analysis of vaginal flora in 30 healthy women and 63 cervical cancer patients, found that the bacterial community composition in the patients' vaginas was disordered, with a decreased relative abundance of beneficial bacteria and an increased relative abundance of opportunistic pathogens, such as Staphylococcus, Clostridium, and Enterobacteriaceae. Further statistical analysis of biochemical indicators in healthy women and patients, and Pearson correlation analysis with the relative abundance of vaginal flora, revealed a positive correlation between the relative abundance of Enterobacteriaceae and CRP in the patients' vaginas (P<0.1). In vitro antitumor cell activity results showed that co-culturing the selected Escherichia coli with cervical cancer tumor cells for 1 hour could produce varying degrees of inhibitory activity against the tumor cells, while inactivated bacteria or the supernatant after bacterial culture did not inhibit tumor cell activity. Subsequent lactate detection and RT-qPCR experiments showed that during the interaction between *E. coli* and tumor cells, the strain could secrete lactate itself or promote the secretion of lactate by tumor cells. Simultaneously, it could significantly increase the gene expression of tumor cell proliferation and migration-related factors. This indicates that the *E. coli* isolated in this invention, in addition to directly inhibiting tumor cell activity, can also alter the tumor microenvironment through autocrine or paracrine systems by secreting metabolites or changing tumor cell metabolic processes, thus affecting its further development. Furthermore, this invention used transmission electron microscopy to observe the morphological changes of *E. coli* after co-incubation with cervical cancer tumor cells, revealing mitochondrial swelling, lysosomal destruction, intracellular degradation, and cell membrane disruption.

[0110] The embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A tumor-inhibiting E. coli 18, with strain number HZ_Ec_18, classified as Escherichia coli HZ_Ec_18, which has been deposited with the China Center for Type Culture Collection, with the accession number CCTCC No: M20251870.

2. Use of the tumor-inhibiting Escherichia coli 18 according to claim 1 for the preparation of a medicament having at least one of the following effects (1) - (2): (1) a medicament for the prevention and / or treatment of a tumor; (2) a medicament having anticancer cell activity; the tumor is selected from the group consisting of cervical cancer, liver cancer, and ovarian cancer; the cancer cell is selected from the group consisting of cervical cancer cell HeLa, cervical cancer cell SiHa, human liver cancer cell HepG2, and human ovarian cancer cell A 2780; the tumor-inhibiting Escherichia coli 18 is present in the form of a living cell.

3. Use according to claim 2, characterized in that, The number of living cells of the tumor-inhibiting Escherichia coli 18 is MOI = 10 - 60.

4. Use according to claim 2, characterized in that, The tumor-inhibiting Escherichia coli 18 is used as the only active ingredient.

5. A pharmaceutical preparation, characterized by, The pharmaceutical preparation comprises a therapeutically effective amount of the tumor-inhibiting Escherichia coli 18 according to claim 1.

6. The pharmaceutical preparation according to claim 5, characterized in that, The pharmaceutical preparation further comprises other pharmaceutically acceptable excipients compatible with the tumor-inhibiting Escherichia coli 18.

7. The pharmaceutical preparation according to claim 5 or 6, characterized in that The pharmaceutical preparation comprises a parenteral administration dosage form and / or a gastrointestinal administration dosage form. The parenteral administration dosage form comprises at least one of an injection administration dosage form, a cavity administration dosage form, a mucosa administration dosage form, and a skin administration dosage form. The gastrointestinal administration dosage form comprises at least one of a tablet, a granule, a capsule, a solution, a powder, an emulsion, and a suspension.

8. The pharmaceutical preparation according to claim 7, characterized in that The gastrointestinal administration dosage form comprises a sustained release preparation.

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